1. Transfect into trp, leu, his mutant yeast cells

2. Select for cells that grow in absence of tryptophan and leucine

3. Plate selected cells on medium lacking histidine

Colony formation

No colony formation

Colony formation

No colony formation

The Yeast Two-Hybrid System Exploits Activator Flexibility to Detect cDNAs That Encode Interacting Proteins

A powerful molecular genetic method called the yeast two-hybrid system exploits the flexibility in activator structures to identify genes whose products bind to a specific protein of interest. Because of the importance of protein-protein interactions in virtually every biological process, the yeast two-hybrid system is used widely in biological research.

This method employs a yeast vector for expressing a DNA-binding domain and flexible linker region without the associated activation domain, such as the deleted GAL4 containing amino acids 1-692 (see Figure 11-17). A cDNA sequence encoding a protein or protein domain of interest, called the bait domain, is fused in frame to the flexible linker region so that the vector will express a hybrid protein composed of the DNA-binding domain, linker region, and bait domain (Figure 11-39a, left). A cDNA library is cloned into multiple copies of a second yeast vector that encodes a strong activation domain and flexible linker, to produce a vector library expressing multiple hybrid proteins, each containing a different fish domain (Figure 11-39a, right).

The bait vector and library of fish vectors are then trans-fected into engineered yeast cells in which the only copy of

M EXPERIMENTAL FIGURE 11-39 The yeast two-hybrid system provides a way of screening a cDNA library for clones encoding proteins that interact with a specific protein of interest. This is a common technique for screening a cDNA library for clones encoding proteins that interact with a specific protein of interest. (a) Two vectors are constructed containing genes that encode hybrid (chimeric) proteins. In one vector (left), coding sequence for the DNA-binding domain of a transcription factor is fused to the sequences for a known protein, referred to as the "bait" domain (light blue). The second vector (right) expresses an activation domain fused to a "fish" domain (green) that interacts with the bait domain. (b) If yeast cells are transformed with vectors expressing both hybrids, the bait and fish portions of the chimeric proteins interact to produce a functional transcriptional activator. In this example, the activator promotes transcription of a HIS gene. One end of this protein complex binds to the upstream activating sequence (UAS) of the HIS3 gene; the other end, consisting of the activation domain, stimulates assembly of the transcription preinitiation complex (orange) at the promoter (yellow). (c) To screen a cDNA library for clones encoding proteins that interact with a particular bait protein of interest, the library is cloned into the vector encoding the activation domain so that hybrid proteins are expressed. The bait vector and fish vectors contain wild-type selectable genes (e.g., a TRP or LEU gene). The only transformed cells that survive the indicated selection scheme are those that express the bait hybrid and a fish hybrid that interacts with it. See the text for discussion. [See S. Fields and O. Song, 1989, Nature 340:245.]

(a) Hybrid proteins

DNA-binding Bait domain domain

Bait hybrid a gene required for histidine synthesis (HIS) is under control of a UAS with binding sites for the DNA-binding domain of the hybrid bait protein. Transcription of the HIS gene requires activation by proteins bound to the UAS. Transformed cells that express the bait hybrid and an interacting fish hybrid will be able to activate transcription of the HIS gene (Figure 11-39b). This system works because of the flexibility in the spacing between the DNA-binding and activation domains of eukaryotic activators.

A two-step selection process is used (Figure 11-39c). The bait vector also expresses a wild-type TRP gene, and the hybrid vector expresses a wild-type LEU gene. Transfected cells are first grown in a medium that lacks tryptophan and leucine but contains histidine. Only cells that have taken up the bait vector and one of the fish plasmids will survive in this medium. The cells that survive then are plated on a medium that lacks histidine. Those cells expressing a fish hybrid that does not bind to the bait hybrid cannot transcribe the HIS gene and consequently will not form a colony on medium lacking histidine. The few cells that express a bait-binding fish hybrid will grow and form colonies in the absence of histidine. Recovery of the fish vectors from these colonies yields cDNAs encoding protein domains that interact with the bait domain.

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